The integrity of a cellular organism is continuously challenged during its lifetime. Internal and external factors, such as toxic compounds and radiation, are a threat to the wellbeing of such organism. Potentially harmful factors comprise factors capable of distorting cellular processes such as the generation of vital biomolecules and/or degradation of such molecules, notably nucleic acids and proteins. For instance, inhibitors of RNA or protein synthesis, transport or turn-over compromise cellular function. Additionally, many internal and external factors are capable of damaging cellular components, such as DNA.
Preservation of an intact genome is of utmost importance to living cellular organisms. However, the integrity of nucleic acids such as DNA is continuously challenged. Cells must overcome endogenous (for instance, metabolic) and exogenous (environmental) threats, as well as the intrinsic instability of chemical bonds in nucleic acid such as DNA itself (e.g., deamination and depurination). For instance, oxidative stress, ultraviolet (UV) light, ionizing radiation (such as X-rays), and numerous chemicals are capable of inducing a wide variety of lesions in DNA. An agent capable of inducing a DNA lesion is called a mutagen. A DNA lesion is defined herein as an alteration of DNA which involves a change in DNA sequence and/or a change in DNA structure. A DNA lesion can, for instance, comprise a DNA (double) strand break and/or an insertion/deletion of at least one nucleotide.
A DNA lesion can affect cellular processes and can have severe consequences for the wellbeing of an organism. Direct effects of DNA lesions at the cellular level comprise inhibition of vital processes like transcription and replication, triggering cell cycle arrest. Accumulation of lesions in DNA above certain thresholds can lead to permanent alterations in the genetic code, replicative senescence and/or to (programmed) cell death. Permanent alterations in the genetic code can, for instance, cause changes in metabolic processes, inborn defects and/or overall functional decline contributing to (premature) aging. Mutations, specifically in proto-oncogenes and tumor suppressor genes, are responsible for tumor initiation and subsequent progression of the multistep process of carcinogenesis. Replicative senescence and cell death can enhance the process of aging.
Potential mutagens are often tested with the widely used Ames test. This test is based upon reversion of mutations in a histidine (his) operon in the bacterium Salmonella typhimurium. The his operon encodes enzymes required for the biosynthesis of the amino acid histidine. Strains with mutations in the his operon are histidine auxotrophs: they are unable to grow without added histidine. Revertants that restore the his+ phenotype will grow on minimal medium plates without histidine.
In the Ames test, the his− mutants are mixed with a potential mutagen and then plated on minimal medium with a very small amount of histidine. The concentration of histidine used is limiting, so after the cells go through several cell divisions, the histidine is used up and the auxotrophs stop growing. However, if the potential mutagen induces his+ revertants during the initial few cell divisions, then each of the resulting revertants will continue to divide and form a colony. The number of colonies produced is proportional to how efficiently a mutagen reverts the original his− mutation.
A disadvantage of the Ames test is that it is unable to detect mutagenic agents that are activated by the eukaryotic (organ or tissue-specific) cellular metabolism (such as the class of p450 enzymes). Although preincubation of the agent to be tested with cellular extracts may partly overcome this limitation, the assay is still unreliable as it utilizes a bacterium to predict effects in a very different organism, such as a mammal, and/or in specific organs or tissues. Next to entire classes of false negative outcomes, also a significant number of false positive results have been obtained with the heterologous procaryotic system. Moreover, the test detects only mutagenic compounds but does not detect agents that have mainly a cytotoxic effect or induce deletions, or other chromosomal aberrations. Finally, the Ames test takes overnight incubation until the result is obtained. This is due to the fact that bacterial growth needs to be awaited.